Primer dimer issue in real time PCR - (Jun/22/2009 )

I just started to work in real time PCR by SYBR green methods, the expected apmplicon is about 80bp, I have got the expected size product in the positive controls and samples, but in melting curve, the Tm is only about 73C, one peak; in 2.5% agrose gel, the size of the product is <100bp;
in the NTC controls, there were no amplification and no peaks in the melting curve. no signals in agrose gel, I want to inquire:
1. based on the above results, could I prove that the amplicon is my product, NOT primer dime? Is it enough that if the NTC shows no signal, then the signal in the samples and the positive controls are products?

2. if it were primer dimer, what should be the dimer size? about 40 bp? (forward plus reverse), could I distinguish it from product (80bp) in 2.5% agrose gel?

3. Another story, by SYBR green method, could I distinuish one base pair mismatch between two templates? If yes, what should I do?

Thanks a lot in advance,

veer2

-veer2-

veer2 on Jun 22 2009, 09:51 AM said:

I just started to work in real time PCR by SYBR green methods, the expected apmplicon is about 80bp, I have got the expected size product in the positive controls and samples, but in melting curve, the Tm is only about 73C, one peak; in 2.5% agrose gel, the size of the product is <100bp;
in the NTC controls, there were no amplification and no peaks in the melting curve. no signals in agrose gel, I want to inquire:
1. based on the above results, could I prove that the amplicon is my product, NOT primer dime? Is it enough that if the NTC shows no signal, then the signal in the samples and the positive controls are products?

2. if it were primer dimer, what should be the dimer size? about 40 bp? (forward plus reverse), could I distinguish it from product (80bp) in 2.5% agrose gel?

3. Another story, by SYBR green method, could I distinuish one base pair mismatch between two templates? If yes, what should I do?

Thanks a lot in advance,

veer2

Is this homework?
Here are the answers to your questions:
1. When you have primer dimers, you usually see in t he dissociation curve a peak with a left shoulder and you'll see a peak with the same Tm in the NTC as well. If you don't see that, then you don't have primer dimers.
2. If your primers are 20nt long, then the primer dimer can be up to 40bp in theory but you could have also concatamers sometimes... You should be able to see it onto a 2.5% gel.
3. I don't think so.
You can read the "performing rq-gene expression" on the Applied Biosystem's website: it will give you the answers you need...

-dnafactory-

Thanks a lot,

-veer2-

Hi veer2,

To answer your 3rd question, it is theoretically possible to distinguish a base pair difference in your amplicon using SYBR Green. The qPCR technique that can do this is called High Resolution Melt (HRM) analysis. It is basically the same as a melt curve only it acquires many more data points during the melting experiment.

To perform HRM you need one of the following qPCR instruments: Roche LightCycler 2.0 or 480, BioRad CFX96 or CFX384, or Qiagen Rotor-Gene Q (these are the only qPCR instruments with native HRM capabilities). The ABI 7500 FAST can also perform HRM, but you need to buy an upgrade to do it.

-ivanbio-

Thanks a lot, dnafactory and ivanbio,

In fact, the reason that I asked the primer dimer problem is: I have 8 genes to be tested by real time PCR. And all the expected amplicon is about 70-80 bps. 7 were good as above (NTC is negative). only one of tested gene which should also have 80bps product by real time PCR, but unfortunately, in NTC there is also one band, the size seems to be the same as the expected product (80bps). And there should be no contamination in NTC. Therefore, I think it should be the primer dimer, but why could not it been distinguished from the true 80 bps product in 2.5% agrose gel. if it is dimer, it should be about 40 bps, or it formed contamers as dnafactory said. I am confused.

As for the distinuishing one base mismatch by SYBR green methods, I read some paper which showed to use 3 steps real time PCR to replace two steps. And set up the anealling temperature exactly 5 centigrade below the Tm of the primer, one base mismatch could be distinuished. I am a little doubtable on it. Has anyone experience??

Thanks a lot

-veer2-

Can you post a print screen of the melting curve, please? You could select the NTC showing the amplicon and one of your samples

-dnafactory-

Hello, thanks a lot, dnafactory
Here I will attached 4 images, first 2 images (Mark 1) are the amplification and melting curve of one gene, the flat line and the mess line in the melting curve is for NTC, on the agrose gel, the samples band is exactly as we expected, and the NTC well shows nothing in the agrose gel, I think it is the product we need. And although the Tm is low as 71 degree, but the product is only 70bps and the Tm prediction of the product is also we predicted.

The next 2 images are for another gene, in the amplificaton curve, the NTC showes small amplification in CT 37, I think it is primer dimer and the melting curve of primer dimer is similar to the product, I could accept this because the expected product is about 70bps, and the primer dimer should be about 40 bps, there is not too much difference between them. But, when I run the 2.5% agrose gel, I could find one very weak band in the NTC well, it is almost the same size as the positive control . It is contamers? or something others? or should I run polyacrimide gel to distinguish it from the product?? I think 40bps and 70bps should be distinguished from each other.

Thanks a lot,

-veer2-

veer2 on Jun 23 2009, 06:40 PM said:

Hello, thanks a lot, dnafactory
Here I will attached 4 images, first 2 images (Mark 1) are the amplification and melting curve of one gene, the flat line and the mess line in the melting curve is for NTC, on the agrose gel, the samples band is exactly as we expected, and the NTC well shows nothing in the agrose gel, I think it is the product we need. And although the Tm is low as 71 degree, but the product is only 70bps and the Tm prediction of the product is also we predicted.

The next 2 images are for another gene, in the amplificaton curve, the NTC showes small amplification in CT 37, I think it is primer dimer and the melting curve of primer dimer is similar to the product, I could accept this because the expected product is about 70bps, and the primer dimer should be about 40 bps, there is not too much difference between them. But, when I run the 2.5% agrose gel, I could find one very weak band in the NTC well, it is almost the same size as the positive control . It is contamers? or something others? or should I run polyacrimide gel to distinguish it from the product?? I think 40bps and 70bps should be distinguished from each other.

This is not your case, where the product comes out and it is much stronger in the "real" sample than in the NTC. Try changing the reagents (i.e. make new dilutions of your primers and use fresh water)

-dnafactory-

Thanks a lot,

veer2

-veer2-

Hi,

The NTC gives me a peak almost every time, I set up a reaction. Currently I am using Roche 480 and analyzing expression of my target gene compared to the housekeeper i.e actin. One other problem is that in my untreated samples (I work with mosquito larvae and expose them to bacteria and use real time PCR to check presence and no. of bacteria) I am getting a low Ct value for my bacterial specific gene, but I am confused as to why would I see any presence of bacteria in my control samples? Also, my target bacterial gene is very specific and should come up positive only if my bacterium is present in the mosquito larvae. Also, is actin expression affected by anything? AS in since actin is my housekeeper gene, should i not be getting the same levels of actin present in day 1 of my treatment and on day3 an day 9? Does it vary with size of the larvae? Is there anyway I can ensure that I have the same amount of actin across all my samples?
Please help..
Thanks,
-Uma